Kenneth E. Kokko

RAPID CLONING OF HIGH AFFINITY HUMAN MONOCLONAL ANTIBODIES AGAINST INFLUENZA VIRUS

Pre-existing neutralizing antibody provides the first line of defence against pathogens in general. For influenza virus, annual vaccinations are given to maintain protective levels of antibody against the currently circulating strains. Here we report that after booster vaccination there was a rapid and robust influenza-specific IgG+ antibody-secreting plasma cell (ASC) response that peaked at approximately day 7 and accounted for up to 6% of peripheral blood B cells. These ASCs could be distinguished from influenza-specific IgG+ memory B cells that peaked 14–21 days after vaccination and averaged 1% of all B cells. Importantly, as much as 80% of ASCs purified at the peak of the response were influenza specific. This ASC response was characterized by a highly restricted B-cell receptor (BCR) repertoire that in some donors was dominated by only a few B-cell clones. This pauci-clonal response, however, showed extensive intraclonal diversification from accumulated somatic mutations. We used the immunoglobulin variable regions isolated from sorted single ASCs to produce over 50 human monoclonal antibodies (mAbs) that bound to the three influenza vaccine strains with high affinity. This strategy demonstrates that we can generate multiple high-affinity mAbs from humans within a month after vaccination. The panel of influenza-virus-specific human mAbs allowed us to address the issue of original antigenic sin (OAS): the phenomenon where the induced antibody shows higher affinity to a previously encountered influenza virus strain compared with the virus strain present in the vaccine. However, we found that most of the influenza-virus-specific mAbs showed the highest affinity for the current vaccine strain. Thus, OAS does not seem to be a common occurrence in normal, healthy adults receiving influenza vaccination.

Systems biology of vaccination for seasonal influenza in humans

Here we have used a systems biology approach to study innate and adaptive responses to vaccination against influenza in humans during three consecutive influenza seasons. We studied healthy adults vaccinated with trivalent inactivated influenza vaccine (TIV) or live attenuated influenza vaccine (LAIV). TIV induced higher antibody titers and more plasmablasts than LAIV did. In subjects vaccinated with TIV, early molecular signatures correlated with and could be used to accurately predict later antibody titers in two independent trials. Notably, expression of the kinase CaMKIV at day 3 was inversely correlated with later antibody titers. Vaccination of CaMKIV-deficient mice with TIV induced enhanced antigen-specific antibody titers, which demonstrated an unappreciated role for CaMKIV in the regulation of antibody responses. Thus, systems approaches can be used to predict immunogenicity and provide new mechanistic insights about vaccines.We used a systems biological approach to study innate and adaptive responses to influenza vaccination in humans, during 3 consecutive influenza seasons. Healthy adults were vaccinated with inactivated (TIV) or live attenuated (LAIV) influenza vaccines. TIV induced greater antibody titers and enhanced numbers of plasmablasts than LAIV. In TIV vaccinees, early molecular signatures correlated with, and accurately predicted, later antibody titers in two independent trials. Interestingly, the expression of Calcium/calmodulin-dependent kinase IV (CamkIV) at day 3 was inversely correlated with later antibody titers. Vaccination of CamkIV −/− mice with TIV induced enhanced antigen-specific antibody titers, demonstrating an unappreciated role for CaMKIV in the regulation of antibody responses. Thus systems approaches can predict immunogenicity, and reveal new mechanistic insights about vaccines.

Inhibition of apical Na+ channels in rabbit cortical collecting tubules by basolateral prostaglandin E2 is modulated by protein kinase C.

UNLABELLED We used the cell-attached patch clamp technique to investigate the interaction of exogenous prostaglandins (PG), intracellular [Ca2+]i, and protein kinase C (PKC) on the high selectivity, 4 pS Na+ channel found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) cultures grown on collagen supports with 1.5 microM aldosterone. Application of 0.5 microM PGE2 to the basolateral membrane decreased mean NP0 (number of channels times the open probability) for apical Na+ channels by 46.5% (n = 9). There was no consistent change in NP0 after apical 0.5 microM PGE2 (n = 12) or after apical or basolateral 0.5 microM PGF2 alpha (n = 8). Release of [Ca2+]i stores with 0.25 microM thapsigargin (n = 7), or activation of apical membrane PKC with apical 0.1 microM 4 beta-phorbol-12-myristate-13-acetate (n = 5) or 10 microM 1-oleyl-2-acetylglycerol (n = 4) also decreased NP0. Depletion of [Ca2+]i stores (0.25 microM thapsigargin pretreatment) (n = 7) or inhibition of apical PKC (100 microM D-sphingosine pretreatment) (n = 8) abolished the inhibitory effects of basolateral PGE2. CONCLUSIONS (a) apical Na+ transport in rabbit CCT principal cells is modulated by basolateral PGE2; (b) the mechanism involves release of IP3-sensitive, [Ca2+]i stores; and (c) Ca(2+)-dependent activation of apical membrane PKC, which then inhibits apical Na+ channels.

Immune responsiveness and protective immunity after transplantation

The growing success of solid organ transplantation poses unique challenges for the implementation of effective immunization strategies. Although live attenuated vaccines have proven benefits for the general population, immunosuppressed patients are at risk for unique complications such as infection from the vaccine because of lack of both clearance and containment of a live attenuated virus. Moreover, while vaccination strategies using killed organisms or purified peptides are believed to be safe for immunosuppressed patients, they may have reduced efficacy in this population. The current lack of knowledge of the basic safety and efficacy of vaccination strategies in the immunosuppressed has limited the development of guidelines regarding vaccination in this population. Recent fears of influenza pandemics and potential attacks by weaponized pathogens such as smallpox heighten the need for increased knowledge. Herein, we review the current understanding of the effects of immunosuppressants on the immune system and the ability of the suppressed immune system to respond to vaccination. This review highlights the need for systematic, longitudinal studies in both humans and nonhuman primates to understand better the defects in innate and adaptive immunity in transplant recipients, thereby aiding the development of strategies to vaccinate these individuals.

Regulation of the amiloride-blockable sodium channel from epithelial tissue.

The first step in net active transepithelial transport of sodium in tight epithelia is mediated by the amiloride-blockable sodium channel in the apical membrane. This sodium channel is the primary site for discretionary control of total body sodium and, therefore, investigating its regulatory mechanisms is important to our understanding of the physiology of fluid and electrolyte balance. Because essentially all of the regulatory sites on the channel are on the intracellular surface, patch clamp methods have proven extremely useful in the electrophysiological characterization of the sodium channel by isolating it from other channel proteins in the epithelial membrane and by allowing access to the intracellular surface of the protein. We have examined three different regulatory mechanisms. (1) Inhibition of channel activity by activation of protein kinase C; (2) activation of the channel by agents which activate G-proteins; and (3) modulation of channel kinetics and channel number by mineralocorticoids. Activation of protein kinase C by phorbol esters or synthetic diacylglycerols reduces the open probability of sodium channels. Protein kinase C can be activated in a physiological context by enhancing apical sodium entry. Actions which reduce sodium entry (low luminal sodium concentrations or the apical application of amiloride) increase channel open probability. The link between sodium entry and activation of protein kinase C appears to be mediated by intracellular calcium activity linked to sodium via a sodium/calcium exchange system. Thus, the intracellular sodium concentration is coupled to sodium entry in a negative feedback loop which promotes constant total entry of sodium. Activation of G-proteins by pertussis toxin greatly increases the open probability of sodium channels. Since channels can also be activated by pertussis toxin or GTP gamma S in excised patches, the G-protein appears to be closely linked in the apical membrane to the sodium channel protein itself. The mechanism for activation of this apical G-protein, when most hormonal and transmitter receptors are physically located on the basolateral membrane, is unclear. Mineralocorticoids such as aldosterone have at least two distinct effects. First, as expected, increasing levels of aldosterone increase the density of functional channels detectable in the apical membrane. Second, contrary to expectations, application of aldosterone increases the open probability of sodium channels. Thus aldosterone promotes the functional appearance of new sodium channels and promotes increased sodium entry through both new and pre-existant channels.

Prostaglandin E2 activates clusters of apical Cl- channels in principal cells via a cyclic adenosine monophosphate-dependent pathway.

UNLABELLED We examined cell-attached patches on principal cells of primary cultured, rabbit cortical collecting tubules. Under basal conditions, apical 9-pS Cl(-)-selective channels were observed in 9% of patches (11/126), and number of channels times open probability (NP0) was 0.56 +/- 0.21. The channel had a linear current-voltage relationship, reversal potential (Erev) near resting membrane potential, a P0 (0.30-0.70) that was independent of voltage, and complicated kinetics (i.e., bursting) at hyperpolarized potentials. NP0 and channel frequency were increased after 30 min of basolateral exposure to 0.5 microM PGE2 (18/56), 10 microM forskolin (23/36), or 0.5 mM dibutyryl cyclic adenosine monophosphate (cAMP) (25/41). Increases in NP0 appeared to be mediated primarily through an increase in the number of observed channels per patch (N), not changes in P0. After these cAMP-increasing maneuvers, N was inconsistent with a uniform distribution of channels in the apical membrane (P < 0.001), but rather the channels appeared to be clustered in pairs. Apical 0.5 microM PGE2 (12/91), apical or basolateral 0.5 microM PGF2 alpha (8/110), or 0.25 microM thapsigargin (releaser of intracellular Ca2+ stores) (7/73) did not increase NP0 or channel frequency. CONCLUSIONS (a) 9-pS Cl- channels provide a conductive pathway for apical membrane Cl- transport across principal cells. (b) Channel activation by basolateral PGE2 is mediated via a cAMP-, but not a Ca(2+)-dependent mechanism. (c) Apical channels are clustered in pairs. (d) With its low baseline frequency and Erev near resting membrane potential, this channel would not contribute significantly to transcellular Cl- flux under basal conditions. (e) However, cAMP-producing agonists (i.e., PGE2, arginine vasopressin) would increase apical Cl- transport with the direction determined by the apical membrane potential.

Regulation of renal epithelial sodium channels

The high selectivity, low conductance, amiloride-blockable, sodium channel of the mammalian distal nephron (i.e. cortical collecting tubule) is the site of discretionary regulation which allows maintainance of total body sodium balance. In order to understand the physiological events that participate in this regulation, we have used the patch-clamp technique which allows us to measure individual Na+ channel currents and permits access to the cytosolic side of the channel-protein as well as its associated regulatory components. Most of our experiments have utilized the A6 amphibian renal cell line, which when grown on permeable supports is an excellent model for the mammalian distal nephron. Different mechanisms have been examined: (1) regulation by hormonal factors such as Anti-Diuretic Hormone (ADH) and aldosterone, (2) regulation by G-proteins, (3) modulation by protein kinase C (PK-C), and (4) modulation by products of arachidonic acid metabolism. Consistent with noise analysis of tight epithelial tissues, ADH treatment increased the number of active channels in apical membrane patches of A6 cells, without any apparent change in the open probability (Po) of the individual channels. Agents that increased intracellular cAMP mimicked the effects of ADH. In contrast, aldosterone was found to act through a dramatic increase in Po rather than through changes in channel density. Inhibition of methylation by deazaadenosine antagonizes the stimulatory effect of aldosterone. In excised inside-out patches GTPγS inhibits channel activity, whereas GDPβS or pertussis toxin stimulates activity suggesting regulatory control by G-proteins. PK-C has been shown to contribute to ‘feed-back inhibition’ of apical Na+ conductance in tight epithelia. Raising luminal bath sodium and therefore intracellular Na+ inhibits sodium channel activity, an effect that is prevented by PK-C inhibitors and mimicked by PK-C agonists. Cyclooxygenase metabolites of arachidonic acid have an inhibitory effect on channel activity. Finally, a possible role for tyrosine kinase as well as membrane cytoskeleton in the regulation of sodium channel function is also suggested.

Cutting Edge: Perforin Down-Regulates CD4 and CD8 T Cell-Mediated Immune Responses to a Transplanted Organ

Perforin mediates target cell apoptosis by CTLs and NK cells. Although perforin expression correlates strongly with acute allograft rejection, perforin-deficient mice reject allografts with the same kinetics as wild-type recipients. In this study, we tested the hypothesis that while perforin is dispensable for acute rejection, it is essential for down-regulating the alloimmune response by inducing the apoptosis of host immune cells. Using a skin transplantation model, we found that perforin-deficient mice are resistant to the induction of allograft acceptance by agents that block T cell costimulation. Failure to induce allograft acceptance in these mice was observed irrespective of whether the alloimmune response was CD4 or CD8 T cell-mediated and could be attributed to defective apoptosis of activated CD4 and CD8 T cells. In contrast, perforin did not influence T cell proliferation. Therefore, perforin is an essential immunoregulatory molecule that may be required for the induction of transplantation tolerance.

Enhanced immunosuppression induced by targeted mutation of cytotoxic T lymphocyte antigen 4-immunoglobulin

Purpose of reviewBlockade of T cell costimulatory pathways has enormous potential as an immunosuppressive strategy to prolong the survival of solid organ transplants. Discovery of the CD28 costimulatory pathway led to the development of both monoclonal antibodies and fusion proteins that interrupt CD28 signaling. Subsequent studies in both animal models and humans demonstrated that although these molecules had promise as immunosuppressive agents for solid organ transplantation, they lacked the desired clinical efficacy. Recent advances in the field of immunobiologics allowed for the development of rationally modified agents, such as LEA29Y (belatacept), that more effectively target costimulatory molecules. This review summarizes the current understanding of the role of CD28 costimulatory blockade as an immunosuppressive strategy in organ transplantation. Recent findingsThis review highlights the strategy used to engineer a fusion protein (LEA29Y, belatacept) from its parent compound (cytotoxic T lymphocyte 4-immunoglobulin) that has higher avidity for CD28 ligands and more potent immunosuppressive effects. SummaryBefore the development of cyclosporine in the early 1980s, the success of transplantation was limited by high rates of rejection and consequent organ loss. Unfortunately, cyclosporine and most subsequently developed immunosuppressive drugs target ubiquitously expressed molecules, causing a myriad of significant side effects unrelated to their primary immunosuppressive effect. Early studies demonstrated complete T cell activation to be critically dependent on signals provided by costimulatory molecules such as CD28 and its ligands B7-1 and B7-2. This provided the impetus for the investigation of agents that block or inhibit these signals with the goal of developing compounds that have less deleterious side effects.

Coexistence of Acute Calcific Periarthritis and Infection

ABSTRACT: Basic calcium phosphate (BCP) crystal deposition around the joints may sometimes lead to an acute inflammatory condition called calcific periarthritis. In this article, the authors describe a 62-year-old man with BCP crystal-induced periarthritis coexisting with an infection. Rheumatoid arthritis and crystal-induced synovitis complicated by infection has been described in the literature. To date, this is the first report of coexistent calcific periarthritis and an infection.